The conversion of a normal cell into one that is cancerous occurs through the acquisition of multiple genetic anomalies. Normally, genetic defects are sensed by checkpoint pathways, which halt cell cycle progression until repairs are made. However, cells which lack these surveillance mechanisms accumulate mutations at a rapid rate. Thus it is not surprising that the most frequently mutated genes in human cancers are those that encode proteins in checkpoint pathways. In S. cerevisiae, one way in which checkpoints prevent cell cycle progression is by blocking activity of the Anaphase Promoting Complex (APC), an E3 ubiquitin ligase. Regulation of the APC is provided by substrate specificity factors that partially account for the specificity and timing of substrate ubiquitination. It is at this level that checkpoint pathways have been shown to exert their inhibitory effect. However, two APC substrates, the mitotic cyclin Clb2p and the spindle-associated protein Ase1P, are known to require the same substrate specificity factor, yet display differential stabilities in late G1. This indicates that other levels of APC regulation exist. Whether these regulatory mechanisms are also affected by checkpoint activation remain to be determined. The goal of this research proposal is to identify novel regulatory mechanisms controlling APC-mediated ubiquitination. Three different approaches will be used, all of which focus upon inactivation of Clb2p proteolysis. Aim 1 addresses possible regulation of Clb2p ubiquitination at the level of substrate, and includes analyses of cis-acting sequences and association with potential stabilizing factors. Aim 2 examines regulation at the level of the ubiquitination machinery, with experiments centered on the APC substrate-specificity factors, Cdc20p and Cdh1p. In the third aim, the question of what regulates Clb2p proteolysis is addressed using an unbiased genetic approach.